Molecular mechanisms of ppGalNAcT catalysis: Previously Tim Fritz, a staff scientist in our group, solved the structures of ppGalNAcTs-1 and 2. Based on these findings, Adina Milac, a visiting fellow and Tim Fritz (in collaboration with Gerard Hummer, NIDDK) performed molecular dynamics simulations of substrate-induced conformational changes of ppGalNAcT-2 (J. Molec. Biol. in press, 2007). Her results reinforced the view that these enzymes employ a single-displacement (SNi) transfer mechanism. Conformational changes in loops located near the active site appear to be crucial for enzymatic activity and the simulations suggested that a conserved tryptophan residue controls the conformational transition of one of these loops. The simulations also suggest that hydrated Mn2+ ion may also play a role in the enzymatic reaction. Previous studies have suggested that the lectin domain of the ppGalNAcTs modulates glycosylation of glycopeptide substrates and underlies the strict glycopeptide specificity of some isoforms (ppGalNAcT-7, -10). Jaya Raman, a visiting fellow and Tim Fritz have examined the activity and glycosylation site preference of lectin domain deletion and swap constructs of the peptide/glycopeptide ppGalNAcT-2 and the glycopeptide ppGalNAcT-10, in collaboration with Tom Gerkin (Case Western Reserve). Her findings indicate that the catalytic domain of ppGalNAcT-10 is unable to recognize non-glycosylated peptide substrate thus accounting for its apparent strict glycopeptide specificity. Further, she demonstrated that the lectin domain of ppGalNAcT-2, actively directs glycosylation site selection. Pre-steady state kinetic measurements show that this effect may be attributable to either lectin domain aided substrate binding or product release after glycosylation. These results highlight the existence of two modes for site selection by ppGalNAcTs: local sequence recognition by structural elements in the catalytic domain that are different between the peptide transferases (ppGalNAcT-2) and the glycopeptide transferases (ppGalNAcT-10) and, the concerted recognition of distal glycosylation events by the lectin domain and local sequence by the catalytic domain. The latter mode would allow the glycosylation of Ser/Thr that occur in sequence contexts that would otherwise be of too low an affinity to be recognized by the catalytic domain alone. A manuscript based on this work will be submitted in late 2007. Tim Fritz has been exploring ways of co-cryrstallizing ppGalNAcTs with glycopeptide and native (apo) substrates. Biological consequences of ablating expression/activity of ppGalNAcTs: We have invested considerable effort in ablating expression of ppGalNAcTs in mice to gain insight into the biological roles of the O-glycans that each isoform initiates. In collaboration with Jamey Marth (HHMI, UCSD) we studied the phenotypes resulting from the deletion of ppGalNAcT-1. Loss of this transferase results in a bleeding disorder which tracks with reduced plasma levels of blood coagulation factors VIII, IX, XI and XII. ppGalNAcTs-1 also supports leukocyte trafficking and residency in inflammatory responses. Animals lacking ppGalNAcTs-1 are also markedly impaired in IgG production coincident with increased germinal center B cell apoptosis and reduced levels of plasma B cells. The Marth lab has communicated a manuscript detailing this work to Molecular Cell Biology. In collaboration with Michael Econs (Univ. Indiana), Tim Fritz has helped to characterize a ppGalNAcT-3 null mouse. In humans, mutation of the isoform results in humoral calcinosis which, among other things, yield high levels of serum phosphate and ectopic calcifications. Dr. Econs is evaluating whether this animal model mimics any of the human phenotype. Yu Guan, a visiting fellow in our lab, is performing additional phenotypic analysis of the ppGalNAc-T1 knockout mouse. She has found evidence of hypertrophy in the heart of 4 mo. old mice. She will be performing functional tests on these mice by MRI (in collaboration with Stasia Anderson of NHLBI) and is also beginning to analyze clotting times in double KO mice to determine if the phenotype observed in ppGalNAcTs-1 nulls is exacerbated when a second member of the glycosyltransferase family is also ablated. Yu is also beginning the characterization of von Willebrand factor isolated from the null animals to ascertain if there are any alterations in glycosylation patterns. Von Willebrand factor forms a complex with Factor VIII and this complex may be disrupted by reduced glycosylation of von Willebrand factor leading to the reduced levels of Factor VIII observed in ppGalNAcT-1 KO mice. Wei Shen, a visiting fellow in our lab, is analyzing the glycosylation patterns of mouse submandibular gland mucin derived from our KO animals. The GalNAc/Ser+Thr ratio of each purified mucin will be determined. If the substitution patterns are altered, our collaborator, Tom Gerkin will map specific O-glycosylation sites from each sample. We are collaborating with two different chemistry groups to identify small molecule inhibitors of ppGalNAcTs (Carolyn Bertozzi, HHMI and UC Berkeley and Suzzane Walker, Harvard Medical School). Wei Shen and Tim Fritz are responsible for the large-scale expression of the ppGalNAcTs that is required to screen the small molecule libraries. Yu Guan and Tim Fritz perform the biological assays on cell lines with candidate inhibitors.